1. Field of the Invention
The invention relates to modulation of gene expression. In particular, the invention relates to modulation of gene expression of the gene encoding DNA methyltransferase, and to modulation of gene expression that is regulated by the enzyme DNA methyltransferase.
2. Summary of the Related Art
Modulation of gene expression has become an increasingly important approach to understanding various cellular processes and their underlying biochemical pathways. Such understanding enriches scientific knowledge and helps lead to new discoveries of how aberrancies in such pathways can lead to serious disease states. Ultimately, such discoveries can lead to the development of effective therapeutic treatments for these diseases.
One type of cellular process that is of particular interest is how the cell regulates the expression of its genes. Aberrant gene expression appears to be responsible for a wide variety of inherited genetic disorders, and has also been implicated in numerous cancers and other diseases. Regulation of gene expression is a complex process, and many aspects of this process remain to be understood. One of the mysteries of this process resides in the fact that while the genetic information is the same in all tissues that constitute a multicellular organism, the expression of functions encoded by the genome varies significantly in different tissues.
In some cases, tissue-specific transcription factors are known to play a role in this phenomenon. (See Maniatis et al., Science 236: 1237-1245 (1987); Ingarham et al., Annual Review of Physiology 52: 773-791 (1990). However, several important cases exist that cannot be readily explained by the action of transcription factors alone. For example, Midgeon, Trends Genet. 10: 230-235 (1994), teaches that X-inactivation involves the inactivation of an allele of a gene that resides on the inactive X-chromosome, while the allele on the active X-chromosome continues to be expressed. In addition, Peterson and Sapienza, Annu. Rev. Genet. 27: 7-31 (1993), describes xe2x80x9cparental imprintingxe2x80x9d, where an allele of a gene that is inherited from one parent is active and the other allele inherited from the other parent is inactive. In both of these cases, both alleles exist in an environment containing the same transcription factors, yet one allele is expressed and the other is silent. Thus, something other than transcription factors must be involved in these phenomena.
Investigators have been probing what type of xe2x80x9cepigenetic informationxe2x80x9d may be involved in this additional control of the expression pattern of the genome. Holliday, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 326: 329-338 (1990) discusses the possible role for DNA methylation in such epigenetic inheritance. DNA contains a set of modifications that is not encoded in the genetic sequence, but is added covalently to DNA using a different enzymatic machinery. These modifications take the form of methylation at the 5 position of cytosine bases in CpG dinucleotides. Numerous studies have suggested that such methylation may well be involved in regulating gene expression, but its precise role has remained elusive. For example, Lock et al., Cell 48: 39-46 (1987), raises questions about whether the timing of hypermethylation and X-inactivation is consistent with a causal role for methylation. Similarly, Bartolomei et al., Genes Dev. 7: 1663-1673 (1993) and Brandeis et al., EMBO J. 12: 3669-3677 (1993), disclose timing/causation questions for the role of methylation in parental imprinting.
Some of the shortcomings of existing studies of the role of DNA methylation in gene expression reside in the tools that are currently available for conducting the studies. Many studies have employed 5-azaC to inhibit DNA methylation. However, 5-azaC is a nucleoside analog that has multiple effects on cellular mechanisms other than DNA methylation, thus making it difficult to interpret data obtained from these studies. Similarly, 5-azadC forms a mechanism based inhibitor upon integration into DNA, but it can cause trapping of DNA methyltransferase (hereinafter, DNA MeTase) molecules on the DNA, resulting in toxicities that may obscure data interpretation.
More recently, Szyf et al., J. Biol. Chem. 267: 12831-12836 (1995), discloses a more promising approach using expression of antisense RNA complementary to the DNA MeTase gene to study the effect of methylation on cancer cells. Szyf and von Hofe, U.S. Pat. No. 5,578,716, discloses the use of antisense oligonucleotides complementary to the DNA MeTase gene to inhibit tumorigenicity. These developments have provided powerful new tools for probing the role of methylation in numerous cellular processes. In addition, they have provided promising new approaches for developing therapeutic compounds that can modulate DNA methylation. The promise of these approaches underscores the importance of developing antisense oligonucleotides complementary to DNA methyltransferase sequences which have optimal activity in inhibiting DNA methyltransferase gene expression. There is, therefore, a need for such optimized antisense oligonucleotides.
The invention provides antisense oligonucleotides complementary to DNA methyltransferase sequences which have optimal activity in inhibiting DNA methyltransferase gene expression. In certain embodiments, the optimized antisense oligonucleotides are characterized by having nucleic acid sequences that provide surprisingly high efficacy. In some of these embodiments, the optimized antisense oligonucleotides according to the invention are further characterized by having distinct chemical modifications which further increase their efficacy. Finally, the invention provides methods for using such antisense oligonucleotides as analytical and diagnostic tools, as potentiators of transgenic plant and animal studies and for gene therapy approaches, and as potential therapeutic agents.
In a first aspect, the invention provides novel optimized antisense oligonucleotides which inhibit the gene expression of DNA MeTase at lower concentration levels of oligonucleotide than previously known oligonucleotides. Such antisense oligonucleotides are complementary to nucleic acid sequences of RNA or double-stranded DNA that encodes DNA MeTase. Preferably, such antisense oligonucleotides contain one or more modified internucleoside linkage and may optionally contain either deoxyribonucleosides, ribonucleosides or 2xe2x80x2-O-substituted ribonucleosides, or any combination thereof. Particularly preferred antisense oligonucleotides according to this aspect of the invention include chimeric oligonucleotides and hybrid oligonucleotides.
In a second aspect, the invention provides a method for investigating the role of DNA MeTase in cellular growth, including the growth of tumor cells. In the method according to this aspect of the invention, the cell type of interest is contacted with an antisense oligonucleotide according to the invention, resulting in inhibition of expression of DNA MeTase in the cell. The antisense oligonucleotides can be administered at different points in the cell cycle, or in conjunction with promoters or inhibitors of cell growth to determine the role of DNA MeTase in the growth of the cell type of interest.
In a third aspect, the invention provides methods for inhibiting tumor growth comprising administering to a mammal, including a human, antisense oligonucleotides according to the invention. In the method according to this aspect of the invention a therapeutically effective amount of an antisense oligonucleotide according to the invention is administered for a therapeutically effective period of time to a mammal, including a human, which has tumor cells present in its body.